1. PROJECT SUMMARY Cigarette smoking causes cardiovascular disease, chronic obstructive pulmonary disease (COPD), and lung cancer. The etiology of diseases caused by exposure to environmental tobacco smoke (ETS) is confounded by the complexity of ETS as a mixture of >4000 chemicals, including direct-acting and metabolically-activated toxicants. While all organisms respond to environmental exposures by regulating gene expression, we know little about the translational mechanisms linking toxicant exposure to cell survival and disease. Here we seek to understand the role of the dozens of modified ribonucleosides in tRNA ? the tRNA epitranscriptome ? in regulating the cell response to ETS exposure. Using a unique computational and analytical platform, we have shown that (1) toxicant exposures cause signature changes in the epitranscriptome of yeast and mammalian cells, and (2) that toxicant-induced reprogramming of the tRNA epitranscriptome regulates protein levels by promoting the selective translation of codon-biased mRNAs from families of stress-response genes in yeast. Our studies in bacteria, yeast and mammalian cells1-7 also show that deficiencies in key epitranscriptomic writer enzymes sensitize cells to killing by specific toxicants due to corrupted translation of stress response proteins. We hypothesize that exposure to the complex mixture of ETS will reprogram the tRNA epitranscriptome to reflect the predominant chemical stressors in ETS and that the altered RNA modifications will regulate cell behavior by selective translation of codon-biased stress response mRNAs. In support of this, we have observed agent-specific epitranscriptome reprogramming in the liver from rats exposed to drugs and toxicants (e.g., arsenic; NTP DrugMatrix), and alkylation- and oxidation-specific signature tRNA modification changes in yeast and human cells. Our epitranscriptomic writer-deficient (Alkbh8-null) mice showed that tRNA modification systems required for translating oxidant-detoxifying selenoproteins are vital to surviving exposure to naphthalene, a P450-activated, oxidant-generating, polycyclic aromatic hydrocarbon (PAH) in ETS. In three aims ranging from in vitro studies in cultured mouse cells to a mouse ETS exposure model, we test the idea that the epitranscriptome and translational regulation play an important role in the cell response to ETS. !